A mechanical filter can be used to remove, filter, collect, concentrate and analyze particles and cells in a variety of fluid medium.
The filtration of cells can aid in the diagnosis of disease. For instance, one of the most important determinants of prognosis and management of cancer is the absence or presence of metastatic dissemination of tumor cells at the time of initial presentation and during treatment. The early spread of tumor cells to lymph nodes or bone marrow is referred to as circulating tumor cells (CTC) when in the peripheral blood. It has been well established that these CTC can be present even in patients who have undergone complete removal of the primary tumor. The detection of CTC has proven to be a useful tool in determining the likelihood of disease progression. Similarly, the ability to detect, collect, or obtain other kinds of cells (e.g., bacterial cells, immune system cells, fetal cells) found in biological fluids by filtration methods will be of great clinical value both in diagnostics and therapeutics.
Further, with respect to blood and CTC, the predominance of the red blood cell can make it difficult to detect or obtain cells found in blood at much lower levels. CTC exist in blood on the order of 1 per 10 billion blood cells. Currently available technologies are inadequate to identify circulating tumor cells with the requisite sensitivity, efficiency and specificity. Existing technologies using magnetic beads, density-gradient centrifugation or polycarbonate filtration for the capturing of tumor cells typically have poor recovery rates and extended processing time in the order of hours.
One way of separating cells is by size. Many research groups have reported cell separation using mechanical filtering. For example, it was demonstrated a weir-type filter with a 3.5 μ gap could isolate leukocytes with a 7% capturing efficiency and >99% erythrocyte rejection (see, Wilding P, et al., Anal Biochem. 257(2):95-100 (1988); Yuen P K, et al., Genome Res. 11(3):405-12 (2001); and Mohamed H, et al., IEEE Trans Nanobioscience 3(4):251-6 (2004). This work indicated an attractive advantage of such devices in obviatin the need to formulate a special buffer condition for separation. More work was, however, warranted to improve the capturing efficiency.
The mechanical filtration method can be applied to circulating tumor cells in blood as these blood cells can substantially differ in size from other major cell types found in blood:
Variability of cell sizes across different cultured tumor cells PBL:peripheral blood lymphocytes(see, Vona G, et al., Am J Pathol. 156(1): 57-63 (2000)Approximate sizeCell CultureTumor Typein μMDA-468BreastNAMCF-7Breast21 ± 2*J82Bladder16 ± 2 T24BladderNAPC3ProstateNALNCaPProstate23 ± 2*Peripheral Blood12 ± 2*Lymphocytes
To handle large sample volumes, as may be required when only a few cancer cells of interest my exist in a large blood volume, a membrane filter needs a large throughput capability. Several groups have demonstrated separation of tumor cells from whole blood with commercially available polycarbonate filters (see, Vona G, et al., Am J Pathol. 160(1):51-8 (2002); Kahn H J, et al., Breast Cancer Res Treat;86(3):237-47 (2004)). However, the existing commercial membrane filters which have been used to mechanically filter blood have low recovery rates. Such filters frequently contain randomly and sparsely distributed holes, with many of them noticeably fused, resulting in large openings that can contribute to lower recovery rates.
Micromachined membrane filters, which have precise geometrical and thickness control, can have better performance. The opening factor can be large and without the defects of fused doublet or triplet holes. Previously, we have demonstrated the manufacture and use of a silicon nitride/parylene membrane filter to separate particles. The particle membrane filters (8×8 mm2) have circular, hexagonal or rectangular through holes. By varying hole dimensions from 6 to 12 μm, opening factors from 4 to 45 % were achieved (see, U.S. Patent Application Publication No. US2001/0019029).
Therefore, there is a need for an improved filtration system which can be used to remove, isolate, capture or detect a cell or other particle in blood or other body fluids. This invention provides for these and other needs.